Significant changes in ethylene synthesis and perception attend many of the developmental transitions experienced within a typical plant life cycle. Such ethylene-dependent processes as shoot elongation, organ abscission, fruit ripening and tissue senescence have long been presumed to be dependent upon the synthesis or activation of ethylene receptors (Trewavas, Physiol. Plant 55: 60–72, 1982). The ability to manipulate ethylene perception was advanced by the identification of ethylene receptors by genetics, positional cloning, and manipulation in transgenic plants (Bleeker et al., Science 241, 1086–1089, 1988; Wilkinson et al., Nature Biotechnology 15, 444–447, 1997; Hua and Meyerowitz, Cell 94, 261–71, 1998).
The characterization of ethylene receptors is leading to refinement of how the processes identified above may be manipulated with precision. The isolation of multiple ethylene receptor candidates and signal transduction components from different plant tissues at disparate stages of development provides a unique opportunity to assess how ethylene promotes and integrates the complex responses of plants to developmental and environmental stimuli. The identification of the roles of specific domains and their modification, the roles of specific receptors in specific tissues, and the ability to replace natural promoters with heterologous promoters all contribute to this refinement.
Several ethylene response genes have been identified in different plants. Among those identified, ETR1 (ethylene response 1) gene has been studied extensively and several ETR1 homologs have been identified and characterized (Sato-Nara et al., Plant Physiol. 119: 321–329, 1999; Lashbrook et al., Plant J. 15 (2): 243–252, 1998; Zhou et al., Plant Mol. Biol. 30: 1331–1338, 1996; Payton et al., Plant Mol. Biol. 31: 1227–1231, 1996). Available data indicate that expression of the ETR1 gene appears to be stage- and tissue-specific and to be regulated during fruit ripening, flower senescence and abscission. The ETR1 gene is expressed in vegetative and reproductive tissues (Zhou et al., Plant Mol. Biol. 30: 1331–1338, 1996) and during fruit development (Sato-Nara et al., Plant Physiol. 119: 321–329, 1999) and it appears to play important roles in plant development and senescence in relation to ethylene perception. In Arabidopsis, as many as five ETR1-like genes have been identified (Lashbrook et al., Plant Journal 15, 243–252, 1998; Theologis, Current Biology 8, 875–878, 1998; Chang et al., Science 262: 539–544, 1993; Hua et al., Science 269: 1712–1714, 1995; Hua et al., Plant Cell 10: 1321–1332, 1998) and they vary significantly in their protein domains. One lacks the carboxy-terminal domain known as the receiver domain while another lacks a critical histidine in the histidine kinase domain though it retains functionality as an ethylene receptor. These variations, particularly the latter, imply that functional differences do exist.
Ethylene insensitivity of plants has been studied by analysis of mutant forms of ETR genes in the homologous species, e.g. ETR1-1 in Arabidopsis (Bleeker et al, Sci. 241: 1086, 1988) and the never ripe mutant in tomato (Lanahan et al., Plant Cell, 6: 521–530, 1994). The ethylene receptor interacts not only with itself in dimerization but must interact with downstream components of the ethylene signal transduction pathway. Specific members of the ethylene receptor pathway may have evolved to interact with specific members of the family of genes comprising the second step in the signal transduction pathway. Because of this protein—protein interaction, use of an ethylene receptor, in its mutated form, of the homologous species and of the same target cell in which ethylene insensitivity is desired is expected to be more efficacious.
Use of an ETR1 gene in its modified form to manipulate the ethylene response in plants may have a great impact on crop yield. Thus, by providing modifications to an ETR1 nucleic acid sequence by substituting, inserting, and/or deleting one or more nucleotides so as to substitute, insert, and/or delete one or more amino acid residues in the protein encoded by the ETR1 nucleic acid, ethylene perception in a plant may be regulated when such a nucleic acid sequence is introduced and expressed in a plant cell. The expression of such a modified ETR1 protein may result in, for example, but not limited to, the retention of more soybean pods and cotton bolls during their development for maturity. To this end, yield will be increased.
In one embodiment an ETR1 nucleic acid, chimeric, mutant thereof, or a portion thereof is placed under the control of an abscission zone (AZ) enhanced promoter, in either a sense or an anti-sense orientation. The promoter could show greater transcriptional enhancement in one AZ or another, for example the pedicel AZ. The expression of this transcript in the AZ could lead to greater retention of reproductive organs, and greater yield.